Elucidating the Molecular Mechanisms of Conformational Switching during Protein Insertion into Membranes

构象转换与蛋白质结构从水溶型转变为 膜插入形式是几个细胞过程中的关键步骤；我们预测和操纵这种形式的能力 切换对人类健康是有益的。构象转换的显著例子包括肿瘤 PH低插入肽(PHLIP)的靶向性、细菌毒素的细胞内进入和膜诱导 激活细胞凋亡调节因子Bcl2家族。虽然这些过程是基本的生物医学 重要的是，需要更好地理解构象转换的机制。具体地说， 调节膜插入的脂质组成和二价阳离子在很大程度上仍未被探索，尽管 越来越多的证据表明了它们在生理上的重要性。我们将检验我们的假设，即血脂的变化 组成、Ca~(2+)和Mg~(2+)浓度以及pH在构象调控中起着关键作用 膜界面。在目标1中，我们将破译预测蛋白质膜的热力学规则。 通过表征质子化和依赖于钙/镁离子的模型多肽的双层分配来研究相互作用。 我们将利用我们的发现来推进现有的基于序列的膜相互作用预测工具 并使其易于应用于对复杂蜂窝环境的更逼真的描述。具体来说， 我们努力预测二价阳离子和可滴定氨基酸残基的pKas变化对 膜界面，这是理解膜依赖的构象重排的关键 这是许多具有生物医学重要性的系统运作的基础。在目标2中，我们将确定 调节pH依赖性和钙/镁依赖性折叠和跨膜过程中的脂质组成 插入PHLIP。这将包括(A)破译钙离子/镁离子和脂质成分对 PHLIP靶向模型膜，(B)用分子方法确定PHLIP-脂质络合物的构象 动力学(MD)模拟和光谱实验，(C)确定单个酸性残基在 PHLIP的构象转换和(D)决定膜中阴离子残基的质子化- 通过核磁共振光谱分析插入了PHLIP。在目标3中，我们将测试在 目的2足以描述PHLIP在含有去混合液的复杂脂类体系中的行为 结构域，以及在哺乳动物细胞中(即，我们将确定pH、二价阳离子、PS和胆固醇的影响 关于PHLIP与细胞膜的相互作用)。

Conformational switching associated with the conversion of a protein structure from a water-soluble to a membrane-inserted form is a critical step in several cellular processes; our ability to predict and manipulate such switching can be beneficial to human health. Notable examples of conformational switching include tumor targeting by the pH Low Insertion Peptide (pHLIP), cellular entry of bacterial toxins, and membrane-induced activation of the Bcl-2 family of apoptotic regulators. While these processes are of fundamental biomedical importance, the mechanism of conformational switching needs to be better understood. Specifically, the role of lipid composition and divalent cations in modulating membrane insertion remains largely unexplored, despite the mounting evidence of their physiological importance. We will test our hypothesis that changes in lipid composition, Ca2+ and Mg2+ concentrations, and pH play key roles in modulating conformational switching on membrane interfaces. In Aim 1, we will decipher the thermodynamic rules for predicting protein-membrane interactions by characterizing the protonation- and Ca2+/Mg2+-dependent bilayer partitioning of model peptides. We will use our findings to advance the existing tools for sequence-based predictions of membrane interactions and make them readily applicable to more realistic descriptions of the complex cellular environment. Specifically, we strive to predict the effects of divalent cations and the changes in pKas of titratable amino acid residues on membrane interfaces, which are critical for understanding membrane-dependent conformational rearrangements that underlie the functioning of many systems of biomedical importance. In Aim 2, we will determine the role of lipid composition in modulating the pH-dependent and Ca2+/Mg2+-dependent folding and transmembrane insertion of pHLIP. This will include (a) deciphering the coupled effect of Ca2+/Mg2+ and lipid composition on pHLIP targeting to model membranes, (b) determining the conformation of pHLIP-lipid complexes by molecular dynamics (MD) simulations and spectroscopic experiments, (c) establish the role of individual acidic residues in the conformational switching of pHLIP and (d) determining the protonation of anionic residues in membrane- inserted pHLIP by NMR spectroscopy. In Aim 3, we will test whether the molecular determinants identified in Aim 2 are sufficient to describe the behaviors of pHLIP in complex lipid systems containing de-mixed liquid domains, and in mammalian cells (i.e., we will determine the effect of pH, divalent cations, PS, and cholesterol on the interaction of pHLIP with cellular membranes).